Protein biosynthesis by the ribosome proceeds in defined phases of initiation, protein elongation, termination, and ribosome recycling (Schmeing 2009). Understanding the molecular mechanism of translation requires high-resolution descriptions of the motions in the ribosome that enable key translational events (Munro 2009; Schmeing 2009; Dunkle 2010). A ratchet-like rotation of the small ribosomal subunit relative to the large ribosomal subunit (Frank 2000) is crucial to the positioning of tRNAs in intermediate—or hybrid—binding sites, in which the 3′-CCA termini and acceptor stems of tRNA advance by one site on the large subunit while the anticodon elements of tRNA remain fixed on the small subunit (Moazed 1989). Binding of tRNAs in hybrid sites is central to mRNA and tRNA movements on the ribosome when they are translocated after peptide bond formation, during termination, and during ribosome recycling (Semenkov 2000; Zavialov 2003). However, the molecular basis for ribosome positioning of tRNAs in hybrid sites has been unclear.
Atomic resolution x-ray crystal structures of the bacterial ribosome with ligands bound have revealed molecular details of conformational rearrangements taking place in the unratcheted ribosome (Schmeing 2009). The first molecular descriptions of intermediate states of ribosome ratchet-like rotation at atomic resolution were provided by x-ray crystal structures of the E. coli 70S ribosome (Zhang 2009), with additional sub-steps proposed based on cryo-EM reconstructions (Fischer 2010). A post-translocation rotated state of the ribosome was recently identified by cryo-EM (Ratje 2010), in a conformation similar to that of the Saccharomyces cerevisiae 80S ribosome in the absence of bound substrates (Ben-Shem 2010).
After the termination of protein synthesis, ribosome recycling is required to free ribosomes from the mRNA transcript to enable further rounds of translation. In bacteria and organelles, ribosome recycling factor (RRF) binds in the tRNA binding cleft of the 70S ribosome at the interface of the large (50S) and small (30S) subunits and interacts with the 50S subunit peptidyl transferase center (PTC) (Lancaster 2002; Agrawal 2004). In so doing, RRF sterically occludes deacylated tRNA binding in the peptidyl-tRNA site (P site, P/P configuration) to favor tRNA positioning in the hybrid peptidyl/exit tRNA binding site (P/E configuration) (FIG. 1A) (Gao 2005; Sternberg 2009). In the P/E configuration, tRNA is bound simultaneously to the P site of the small (30S) subunit and to the E site of the large (50S) subunit (Moazed 1989). Binding of the GTPase elongation factor-G (EF-G) to the RRF-ribosome complex and subsequent GTP hydrolysis lead to the dissociation of ribosomal subunits (Savelsbergh 2009).
Crystallization of biomolecules generally proceeds by empirical exploration of potential crystallization conditions and is a slow and arduous process, even when considerable structural information may already be available. For example, if the biomolecule of interest is known to exist in, or to sample, multiple conformational states, as is the case with the ribosome during the defined phases of initiation, protein elongation, termination, and ribosome recycling, and one of those states is thermodynamically favored, it may be very difficult to find suitable crystallization conditions from which to crystallize the biomolecule into a different conformational state. Hence, providing a method to rapidly vary solution conditions while simultaneously sampling the conformational states of the biomolecule across those conditions, would allow one to rapidly ascertain the parameters important for maintaining the alternate conformational state. Such an ability would be invaluable for optimizing crystallization conditions to favor a desired conformation of a biomolecule because one can alter the energy landscape so that the “normally transient” conformation becomes favored under the solution conditions identified through screening. As shown herein, single-molecule, fluorescence resonance energy transfer (smFRET) imaging methods are adaptable to rapid screening techniques and can provide the type of information needed to optimize crystallization conditions to stabilize a specific conformation of a biomolecule.
For example, prior to the present work, high-resolution (e.g., atomic resolution) structures of all functionally relevant ribosome complexes were found to occupy the classical, unrotated conformation. The use of smFRET imaging methods showed the likely reason for this empirical observation is that the bacterial ribosome is thermodynamically more stable in this configuration. Thus, to determine the structure of the hybrid, rotated ribosome conformation, smFRET imaging methods were used to establish experimental conditions in which functional ribosome complexes were “stabilized” in the desired conformation (e.g., hybrid tRNA, “unlocked state” configuration). These conditions, which were rapidly determined by smFRET imaging, and enabled crystallization of the ribosome in this rotated configuration.
Hence, single-molecule imaging techniques can be used to rapidly optimize solution conditions, screen including the presence or absence of one or more ligands, if applicable, or even screen mutants, to generate crystals of a biomolecule in a desired conformation.